Camellia Sinensis Mouthwashes in Oral Care: a Systematic Review

Herbal products are increasingly growing in the oral care market. Some of the related herbal compounds in this field have considerable clinical evidence for use in mouthwashes in their background. Camellia sinensis or tea plant has attracted numerous researchers of dentistry and pharmaceutical sciences, in recent years, for its biologic and medicinal properties. The effects such as anti-septic, anti-oxidative, and anti-inflammatory activities have made this plant a suitable candidate for preparation of mouthwashes. In this systematic review, we tried to find, evaluate, and categorize the sparse evidence in medical literature about Camellia sinensis mouthwashes. We explored three scientific databases with keywords including tea, dental care, Camellia sinensis, and mouthwashes and found 69 relevant studies including 41 randomized controlled trials (RCTs), which are generally proposing anti-microbial, anti-plaque, and analgesic indications for these tea formulations. Considering the main trend in clinical evidence and favorable safety profile, Camellia sinensis products are able to act as antiseptic, anti-plaque, and anti-inflammatory agents and can be used as useful mouthwashes in the future clinical studies and practice.


Introduction
Herbal agents have been extensively entered into oral care products, in recent years. These compounds have interesting medical and physiochemical characteristics.
Nowadays lots of manufacturers are using herbal ingredients in their products to provide additional therapeutic characteristics. Among these herbs, Camellia sinensis or tea plant has a unique phytochemical and pharmacological profile, which makes it a perfect candidate for use as active constituent in oral mouthwashes [1].
Numerous compounds have been discovered in tea plant with therapeutic effects. These beneficial properties are mostly connected to polyphenols, which usually account for 30% of the dry weight of the solid materials in the brewed green tea [2]. Anti-cancer effect of polyphenols of green tea has been evaluated in many in vitro studies [3][4][5][6][7][8]. An in vitro study on epigallocatechin gallate (EGCG), an active ingredient found in Camellia sinensis, showed that EGCG decreases the expression of phosphorylated epidermal growth factor receptors in oral cancer cells, as well as non-oral ones [7]. Cell cycle analysis on oral squamous cell carcinoma cells shows that EGCG induces G1 phase arrest and increases occurrence of apoptosis among tumor cells [9]. It has also been reported that green tea polyphenols possess anticolorectal cancer activity, by altering intestinal microbiota and intestinal colonization of oral cavity bacteria that are associated with gastrointestinal malignancies [10]. Studies have also revealed anti-angiogenic effects for green tea catechins, which can suppress tumor growth [11][12][13][14]. Cardio-protective [15][16], cholesterol lowering [17][18][19][20], anti-hypertensive [21][22][23], and antidiabetic [24][25] properties of polyphenols has also been indicated in many clinical studies and literature reviews.
Neurological studies indicate that green tea polyphenols act as antioxidant compounds, which have neuro-protective benefits and modulatory function on intracellular signaling, and cell survival/death genes; hence, they can be useful in prevention and treatment of neurodegenerative diseases [26][27][28][29][30]. Antimicrobial, antiinflammatory, and anti-nociceptive properties of Camellia sinensis extract have been the subject of many studies, as well [2,[31][32][33][34][35][36]. Green tea antibacterial function has been related to its EGCG and other compounds through damaging the bacterial cell membrane [37], inhibitory effect against gyrase enzyme, and destroying cytoplasmic membrane of bacterium [38]. Antiinflammatory function of EGCG is mainly exerted by down-regulation of Cyclooxygenase-2 via inhibition of interleukin-1b-dependent pro-inflammatory signal transduction and interleukin-6, interleukin-8 and tumor necrosis factor-α gene expression at inflammation sites [39][40]. These characteristics have attracted the scientists for substantial evaluations in local application of tea products. Accordingly, dental researches have also focused on tea-related formulations, in mouthwash forms, for treatment of several oral diseases [41][42][43].
In this review, we tried to collect and categorize the potential applications of Camellia mouthwashes in different dentistry fields to demonstrate the most evidencebased indications with their advantages and disadvantages.

Search Strategy
In order to provide a systematic review with the highest accordance to Preferred Reporting Items for Systematic Reviews and Meta-Analyses (PRISMA) guidelines, besides a comprehensive and acceptably relevant data pool, we searched three highly accredited databases including PubMed, Cochrane library, and Google Scholar. We gave a priority to PubMed database, due to its privileges and scientific characteristics for systematic searching.

Inclusion criteria
The inclusion criteria involved journal articles, those with titles including "green tea" or "tea" or "Camellia sinensis" and "mouthwash" or "mouth rinse" or "gargle" in the field of dentistry. Our exclusion criteria were non-English-abstract articles and those focused on adverse effects or staining capabilities of green tea products. We explored PubMed database with MeSH terms, "tea", and "mouthwashes" at the first step. We also tried "Camellia sinensis" and "mouthwashes" for MeSH  : Flow diagram depicting the study selection and categorization process for "Camellia sinensis/tea" and "mouthwashes" search results gathering and categorization of the evidence. Therefore, performing a meta-analysis was relinquished, intentionally.

Discussion
In this part, we classified the beneficial effects of different tea-based local oral formulations based on the type of scientific evidence and potential clinical indications.
These involve both basic experimental studies with clinical implications and clinical trials.

In vitro and animal studies
Anti-bacterial properties were the major focus of nu-  [46].
Anti-fungal property of green tea has been the subject of many in vitro studies, as well. Sitheeque et al. [47] showed Candida glabrata is the most sensitive species of Candida to tea polyphenols followed by Candida parapsilosis and Candida albicans (C. albicans). Since being the most common fungal species found in oral cavity, anti-fungal activity of green tea is mostly evaluated against C. albicans [48]. It is suggested that the EGCG found in green tea acts as an antifolate compound, which disturbs metabolism of folic acid in C. albicans [49]. Another study showed that polyphenolic compounds found in green tea extract, caused 75% reduction in viable C. albicans cells during biofilm formation. This research proposed that polyphenols anti-fungal activity is exerted via reduction of proteasomal activity in C.albicans cells [50]. Another study conducted by Yadegari et al. [51], demonstrated that inhibitory effect of EGCG against C. albicans is time dependent. The results also showed that EGCG could be effective on fluconazole-resistant C. albicans. It has also been stated that green tea aqueous extract can be useful against C. albicans colonies growing on acrylic resin and polyvinyl chloride surfaces used in prosthetic dentures and orthodontic appliances [52][53]. In a study conducted by Mollashahi et al. [54], on tooth substrate of extracted premolar teeth, it was implied that green tea extract has the potential to be used as an irrigating agent in endodontic treatments due to its anti-fungal properties. However, the researchers of the study suggested evaluating its biocompatibility and safety before using it as an irrigating solution in clinical settings. Another study in the department of biotechnology in Graphic Era University, India, investigated synergistic activity of green tea extract and fluconazole, amphotericin B and copper sulphate as a combinational therapy against C.
albicans. The results proved a synergic effect that can be efficient against C. albicans and implied that the combinational therapy does not show cytotoxic activity on healthy cells [55].
Some in vivo experiments have studied green tea's anti-inflammatory function. An animal study on type 1 diabetic rats showed green tea extract minimizes expression of receptor activator of nuclear factor kappa-Β ligand and tumor necrosis factor-alpha, consequently decelerating alveolar bone resorption compared to placebo group [56]. Another animal observation on albino rats using green tea extract, illustrated a significant reduction in nicotine-induced damage, in terms of inflammatory cell infiltrates in buccal mucosa [57]. Such observations proposed the potential benefits of tea mouthwash preparations in clinical application. However, there are some controversial findings. Regarding the wound healing, an animal study failed to show a significant effect for green tea formulation [58].

Clinical trials and RCTs
Screening and assessment of clinical trials including RCTs in this review revealed a growing trend for utilization of Camellia products in dentistry clinical practice. Almost every group of authors examined a new and divergent application for such formulations, which are categorized in the following sections. Specifications of the RCTs are summarized in Table 1.

Randomized controlled trials
At first, we explored the real-life indications of green tea mouthwashes in RCTs in order to provide a more accurate insight toward clinical applications of these products.

Antiseptic mouthwashes
As mentioned before, microbial regulation is the main purpose of using tea rinses in multiple studies. In a clinical trial on 30 healthy volunteers, gargling with two commercial tea extract solutions for 60 seconds resulted in significant decrease in microbial counts of expectorated oral rinses. Compared to a synthetic antiseptic mouthwash, those herbal oral rinses showed prolonged activity [59]. In addition to teeth and oral cavity, the effect of mouthwashes on devices and surgical derbies has also been assessed. In another RCT, performed on 18 patients with unerupted maxillary third molars with extraction indication, researchers tried to evaluate the anti-microbial and anti-adherence activity of different mouthwashes on suture materials after the surgery. Participants were equally divided in three groups of Camellia sinensis, Calendula officinalis, or CHX mouthwash users for one week. Compared to no intervention, as a control, all solutions were effective to reduce the number of adhered microorganisms, but the only significant difference was observed for CHX [60]. The same results was found in an RCT done by Prabakar et al. [61], in which they showed that CHX and green tea  [70]. In another experiment, the same treatment protocol was implemented on intubated patients admitted to intensive care unit for evaluation of bacterial colonies in their throat culture.
Results of this study showed green tea and CHX had the same efficacy in reducing pharyngeal bacterial load [71]. A crossover RCT in Mashhad University of Medical Sciences evaluated incidence of alveolar osteitis after using green tea mouthwash following third molar surgery. The intervention resulted in no clinically significant difference for the mouthwash users compared to control group [72].
Besides local cleansing effects, green tea gargle has also been evaluated for prevention of airborne systemic infections. Ide et al. [73] randomized 757 high school children into green tea and tap water gargle three times daily during the influenza epidemic season. Despite the fact that no significant advantage was observed for green tea for flu prevention, authors stated that modification of the non-blinded design, controlling for better adherence rates in participants, and utilization of better placebos or diagnostic tools would probably reveal more promising results in the future studies. In a secondary analysis of these results using Bayesian estimation, the authors found that green tea gargling has a slight superiority over water gargling for that indication [74].
A clinical comparative evaluation between green tea mouthwash and green tea gel showed no significant difference in their antibacterial potency [75]. Considering CHX as an old and well-known oral disinfectant, tea mouthwashes have shown relatively acceptable antimicrobial effects without any of the complications associated with synthetic chemicals, including the adverse effects.

Anti-plaque mouthwashes
Dental hygiene has also been evaluated in tea mouthwash trials. Potential anti-plaque effects have been in- showed that use of tea mouthwash was more effective in terms of plaque-score. In addition, it improved gingival and oral hygiene status. There was no placebo group in this trial [76]. In a larger RCT on 110 industrial male workers with mean plaque, index (PI) of 1.5 and gingival index (GI) of 1, participants were divided into green tea extract and placebo mouthwash groups. They were instructed to use the rinses twice daily, 30 seconds each time for 28 days. Results revealed that the PI and GI were significantly decreased in the green tea group [77].
Another comparative RCT conducted by Romoozi et al. [78], demonstrated that besides being as effective as CHX mouthwash in improving PI and GI in patients with plaque-induced gingivitis, tea mouthwash has the privilege of no dental staining which is a common side effect of CHX. Moreover, four RCTs with similar protocols have also verified the effectiveness of green tea mouthwash at the levels of CHX in improving plaque and gingival indices in patients with plaque-induced gingivitis [79][80][81][82]. On the other hand, in an RCT conducted in 2018 by Nagar et al. [83], 30 dental students whom were divided into CHX, green tea, and white tea groups were asked to use the prescribed mouthwashes for 10 days. According to the results, in spite of significant improvements in gingival and plaque indices by herbal formulations, they were less effective compared to CHX. In addition, in a single-blind, one-operator, three-period, three-treatment clinical study with a randomized Latin square cross-over design on 30 volunteers, no significant reduction in plaque surface area was detected for tea mouthwash compared to a synthetic commercial product [84].
Regarding the saliva acidity, an RCT showed green tea mouthwash to be more effective in increasing salivary pH than CHX and sodium fluoride mouthwashes [85]. In comparison with probiotics, green tea mouth rinse demonstrated similar success in increasing salivary pH, which can be beneficial in prevention of enamel demineralization [86].
Plaque control and oral hygiene maintenance has always been a major concern in orthodontic patients. In an RCT on 30 orthodontic patients with fixed orthodontic appliances, anti-plaque efficacy of CHX (as a routine prescribed mouth rinse in orthodontic treatments) and green tea mouthwash was compared. This study showed superiority in plaque reduction for green tea over CHX and Listerine mouthwash [87]. Contrarily, in another RCT, during fixed orthodontic treatment green tea exerted significant plaque reduction, while it was not as efficient as CHX [88].
C-reactive protein and alkaline phosphatase are considered two nonspecific inflammatory markers, which rise in peripheral blood circulation and gingival crevicular fluid as a sign of active periodontitis [89][90]. An RCT on 45 patients with mild to moderate localized chronic periodontitis showed that administration of green tea 5% mouthwash for two weeks significantly reduced these biomarkers in blood circulation in chronic periodontitis [91]. Lamba et al. [92] have also proposed that using green tea mouthwash after the phase I of periodontal treatments (Oral hygiene education, scaling, and root planning) had significantly higher effect in reducing the total leukocyte count, especially neutrophil count, compared to placebo. Anti-plaque effect of tea rinses is mainly related to its anti-microbial effect but presence of other mechanisms cannot be ruled out. It is almost a proven fact that these products can be efficiently used for plaque fighting.

Painkiller mouthwashes
Oral pain is a very common manifestation of numerous clinical presentations and pathologies in dental patients. showed that lowest plaque score was yielded by herbal product followed by CHX and then the placebo was ranked, with statistically significant differences [96]. In another clinical study, combination of tea extract with sodium lauryl sulfate in a mouthwash produced a greater anti-bacterial potency than the extract alone [59]. A randomized clinical evaluation between "green tea" and "green tea with Xylitol" mouthwashes, regarding their efficacy against salivary S. mutans and Lactobacillus colonies, findings demonstrated that addition of Xylitol to green tea extract had better antibacterial efficacy compared to green tea extract alone [97]. Some studies have indicated that combining green tea and Aloe vera extracts creates a more potent antimicrobial herbal mouthwash. An RCT on patients suffering from periodontitis showed combination of Camellia sinensis and Aloe vera extract has been highly effective in improvement of gingival indices [98]. Another comparative clinical trial between Aloe Vera-Green Tea, CHX, and matrica mouthwashes displayed that Aloe Vera-Green Tea mouthwash was as effective as CHX in reducing PI, GI, and bleeding on probing index and exerts more antiplaque potency than matrica mouthwash [99]. In a quasi-experimental study, 81 orally intubated patients hospitalized in intensive care unit of a medical center in Taiwan were divided into three oral care pro-tocol groups: green tea, boiled water, and control group.
This study revealed irrigation with green tea extract in orally intubated patients could minimize mucosal changes and dental plaque accumulation and would be beneficial in improving their oral health status [102].
The efficacy of green tea mouthwash in management of halitosis has also been evaluated. interesting issue in this context is the noticeable safety profile of these formulations, which is an outstanding advantage for this herbal mouthwash over synthetic chemical ones.

Acknowledgments
There was no source of support or fund for preparation of this manuscript. All authors have accepted responsibility for the entire content of this submitted manuscript and approved submission.